Insight into post-transcriptional regulatory mechanisms of bacterial gene expression will be sought through the study of a novel paradigm in global regulation, the carbon storage regulatory system (Csr) of Escherichia coIi. Csr includes an RNA-binding protein, CsrA, which is a potent modulator of specific mRNA stability, and a non-coding RNA molecule, CsrB. CsrB forms a ribonucleoprotein complex with CsrA and antagonizes its activity. It is hypothesized that CsrB RNA abrogates repression by competing with regulated mRNAs for binding to CsrA, essentially "titrating-out" the CsrA protein. In E. coli, CsrA affects metabolism, physiology and cell surface properties on a broad scale, repressing certain genes expressed during the transition from the exponential phase of growth into stationary phase and activating various genes expressed in the exponential phase. Homologs of csrA are widely-distributed among eubacteria and repress the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will also provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections. Specific aims of this proposal are: 1) To further define the molecular mechanism by which CsrA facilitates mRNA decay. This will include determination of the sequence and structural requirements for mRNA recognition, and the Potential regulatory role(s) of CsrA in translational inhibition and/or in the facilitation of endonucleolytic cleavage of target transcripts. 2) We will assess the molecular mechanism and regulatory role of CsrB in antagonizing the activity of CsrA. The effects of CsrB on CsrA-regulated mRNA decay will be determined in vivo, and the isolated CsrA-CsrB complex will be characterized to determine the CsrB RNA segments involved in CsrA binding. 3) The means by which the Csr system mediates response to environmental and physiological conditions will be explored by determining the genetic, physiological and temporal factors which influence CsrA and CsrB levels and csrA and csrB gene expression. The long-range basic objectives of our investigations in this area are to fully understand the regulatory components and genetic circuitry, molecular mechanisms, and regulatory/biological functions of the Csr system.